Rozana Rosli

Oil palm genome sequence reveals divergence of interfertile species in Old and New worlds

Oil palm is the most productive oil-bearing crop. Although it is planted on only 5% of the total world vegetable oil acreage, palm oil accounts for 33% of vegetable oil and 45% of edible oil worldwide, but increased cultivation competes with dwindling rainforest reserves. We report the 1.8-gigabase (Gb) genome sequence of the African oil palm Elaeis guineensis, the predominant source of worldwide oil production. A total of 1.535 Gb of assembled sequence and transcriptome data from 30 tissue types were used to predict at least 34,802 genes, including oil biosynthesis genes and homologues of WRINKLED1 (WRI1), and other transcriptional regulators, which are highly expressed in the kernel. We also report the draft sequence of the South American oil palm Elaeis oleifera, which has the same number of chromosomes (2n = 32) and produces fertile interspecific hybrids with E. guineensis but seems to have diverged in the New World. Segmental duplications of chromosome arms define the palaeotetraploid origin of palm trees. The oil palm sequence enables the discovery of genes for important traits as well as somaclonal epigenetic alterations that restrict the use of clones in commercial plantings, and should therefore help to achieve sustainability for biofuels and edible oils, reducing the rainforest footprint of this tropical plantation crop.

The oil palm SHELL gene controls oil yield and encodes a homologue of SEEDSTICK

A key event in the domestication and breeding of the oil palm Elaeis guineensis was loss of the thick coconut-like shell surrounding the kernel. Modern E. guineensis has three fruit forms, dura (thick-shelled), pisifera (shell-less) and tenera (thin-shelled), a hybrid between dura and pisifera. The pisifera palm is usually female-sterile. The tenera palm yields far more oil than dura, and is the basis for commercial palm oil production in all of southeast Asia. Here we describe the mapping and identification of the SHELL gene responsible for the different fruit forms. Using homozygosity mapping by sequencing, we found two independent mutations in the DNA-binding domain of a homologue of the MADS-box gene SEEDSTICK (STK, also known as AGAMOUS-LIKE 11), which controls ovule identity and seed development in Arabidopsis. The SHELL gene is responsible for the tenera phenotype in both cultivated and wild palms from sub-Saharan Africa, and our findings provide a genetic explanation for the single gene hybrid vigour (or heterosis) attributed to SHELL, via heterodimerization. This gene mutation explains the single most important economic trait in oil palm, and has implications for the competing interests of global edible oil production, biofuels and rainforest conservation.

Exploiting an oil palm EST database for the development of gene-derived SSR markers and their exploitation for assessment of genetic diversity.

A total of 5,521 expressed sequence tags (ESTs) from oil palm were used to search for type and frequency of simple sequence repeat (SSR) markers. Dimeric repeat motifs appeared to be the most abundant, followed by tri-nucleotide repeats. Redundancy was eliminated in the original EST set, resulting in 145 SSRs in 136 unique ESTs (114 singletons and 22 clusters). Primers were designed for 94 (69.1%) of the unique ESTs (consisting of 14 consensus and 80 singletons). Primers for 10 EST-SSRs were developed and used to evaluate the genetic diversity of 76 accessions of oil palm originating from seven countries in Africa, and the standard Deli dura population. The average number of observed and effective alleles was 2.56 and 1.84, respectively. The EST-SSR markers were found to be polymorphic with a mean polymorphic information content value of 0.53. Genetic differentiation (FST) among the populations studied was 0.2492 indicating high level of genetic divergence. Moreover, the UPGMA (unweighted pair-group method with arithmetic mean) analysis revealed a strong association between genetic distance and geographic location of the populations studied. The germplasm materials exhibited higher diversity than Deli dura, indicating their potential usefulness in oil palm improvement programmes. The study also revealed that the populations from Nigeria, Congo and Cameroon showed the highest diversity among the germplasm evaluated in this study. The EST-SSRs further demonstrated their worth as a new source of polymorphic markers for phylogenetic analysis, since a high percentage of the markers showed transferability across species and palm taxa.

SSR mining in oil palm EST database: application in oil palm germplasm diversity studies

This study reports on the detection of additional expressed sequence tags (EST) derived simple sequence repeat (SSR) markers for the oil palm. A large collection of 19243 Elaeis guineensis ESTs were assembled to give 10258 unique sequences, of which 629 ESTs were found to contain 722 SSRs with a variety of motifs. Dinucleotide repeats formed the largest group (45.6%) consisting of 66.9% AG/CT, 21.9% AT/AT, 10.9% AC/GT and 0.3% CG/CG motifs. This was followed by trinucleotide repeats, which is the second most abundant repeat types (34.5%) consisting of AAG/CTT (23.3%), AGG/CCT (13.7%), CCG/CGG (11.2%), AAT/ATT (10.8%), AGC/GCT (10.0%), ACT/AGT (8.8%), ACG/CGT (7.6%), ACC/GGT (7.2%), AAC/GTT (3.6%) and AGT/ACT (3.6%) motifs. Primer pairs were designed for 405 unique EST-SSRs and 15 of these were used to genotype 105 E. guineensis and 30 E. oleifera accessions. Fourteen SSRs were polymorphic in at least one germplasm revealing a total of 101 alleles. The high percentage (78.0%) of alleles found to be specific for either E. guineensis or E. oleifera has increased the power for discriminating the two species. The estimates of genetic differentiation detected by EST-SSRs were compared to those reported previously. The transferability across palm taxa to two Cocos nucifera and six exotic palms is also presented. The polymerase chain reaction (PCR) products of three primer-pairs detected in E. guineensis, E. oleifera, C. nucifera and Jessinia bataua were cloned and sequenced. Sequence alignments showed mutations within the SSR site and the flanking regions. Phenetic analysis based on the sequence data revealed that C. nucifera is closer to oil palm compared to J. bataua; consistent with the taxanomic classification.

The oil palm VIRESCENS gene controls fruit colour and encodes a R2R3-MYB

Oil palm, a plantation crop of major economic importance in Southeast Asia, is the predominant source of edible oil worldwide. We report the identification of the VIRESCENS (VIR) gene, which controls fruit exocarp colour and is an indicator of ripeness. VIR is a R2R3-MYB transcription factor with homology to Lilium LhMYB12 and similarity to Arabidopsis PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1). We identify five independent mutant alleles of VIR in over 400 accessions from sub-Saharan Africa that account for the dominant-negative virescens phenotype. Each mutation results in premature termination of the carboxy-terminal domain of VIR, resembling McClintock’s C1-I allele in maize. The abundance of alleles likely reflects cultural practices, by which fruits were venerated for magical and medicinal properties. The identification of VIR will allow selection of the trait at the seed or early-nursery stage, 3-6 years before fruits are produced, greatly advancing introgression into elite breeding material.

Seqping: gene prediction pipeline for plant genomes using self-training gene models and transcriptomic data

BackgroundGene prediction is one of the most important steps in the genome annotation process. A large number of software tools and pipelines developed by various computing techniques are available for gene prediction. However, these systems have yet to accurately predict all or even most of the protein-coding regions. Furthermore, none of the currently available gene-finders has a universal Hidden Markov Model (HMM) that can perform gene prediction for all organisms equally well in an automatic fashion.ResultsWe present an automated gene prediction pipeline, Seqping that uses self-training HMM models and transcriptomic data. The pipeline processes the genome and transcriptome sequences of the target species using GlimmerHMM, SNAP, and AUGUSTUS pipelines, followed by MAKER2 program to combine predictions from the three tools in association with the transcriptomic evidence. Seqping generates species-specific HMMs that are able to offer unbiased gene predictions. The pipeline was evaluated using the Oryza sativa and Arabidopsis thaliana genomes. Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis showed that the pipeline was able to identify at least 95% of BUSCO’s plantae dataset. Our evaluation shows that Seqping was able to generate better gene predictions compared to three HMM-based programs (MAKER2, GlimmerHMM and AUGUSTUS) using their respective available HMMs. Seqping had the highest accuracy in rice (0.5648 for CDS, 0.4468 for exon, and 0.6695 nucleotide structure) and A. thaliana (0.5808 for CDS, 0.5955 for exon, and 0.8839 nucleotide structure).ConclusionsSeqping provides researchers a seamless pipeline to train species-specific HMMs and predict genes in newly sequenced or less-studied genomes. We conclude that the Seqping pipeline predictions are more accurate than gene predictions using the other three approaches with the default or available HMMs.

Elaeis oleifera genomic-SSR markers: exploitation in oil palm germplasm diversity and cross-amplification in arecaceae.

Species-specific simple sequence repeat (SSR) markers are favored for genetic studies and marker-assisted selection (MAS) breeding for oil palm genetic improvement. This report characterizes 20 SSR markers from an Elaeis oleifera genomic library (gSSR). Characterization of the repeat type in 2000 sequences revealed a high percentage of di-nucleotides (63.6%), followed by tri-nucleotides (24.2%). Primer pairs were successfully designed for 394 of the E. oleifera gSSRs. Subsequent analysis showed the ability of the 20 selected E. oleifera gSSR markers to reveal genetic diversity in the genus Elaeis. The average Polymorphism Information Content (PIC) value for the SSRs was 0.402, with the tri-repeats showing the highest average PIC (0.626). Low values of observed heterozygosity (Ho) (0.164) and highly positive fixation indices (Fis) in the E. oleifera germplasm collection, compared to the E. guineensis, indicated an excess of homozygosity in E. oleifera. The transferability of the markers to closely related palms, Elaeis guineensis, Cocos nucifera and ornamental palms is also reported. Sequencing the amplicons of three selected E. oleifera gSSRs across both species and palm taxa revealed variations in the repeat-units. The study showed the potential of E. oleifera gSSR markers to reveal genetic diversity in the genus Elaeis. The markers are also a valuable genetic resource for studying E. oleifera and other genus in the Arecaceae family.

Analyses of Hypomethylated Oil Palm Gene Space

Demand for palm oil has been increasing by an average of ∼8% the past decade and currently accounts for about 59% of the worlds vegetable oil market. This drives the need to increase palm oil production. Nevertheless, due to the increasing need for sustainable production, it is imperative to increase productivity rather than the area cultivated. Studies on the oil palm genome are essential to help identify genes or markers that are associated with important processes or traits, such as flowering, yield and disease resistance. To achieve this, 294,115 and 150,744 sequences from the hypomethylated or gene-rich regions of Elaeis guineensis and E. oleifera genome were sequenced and assembled into contigs. An additional 16,427 shot-gun sequences and 176 bacterial artificial chromosomes (BAC) were also generated to check the quality of libraries constructed. Comparison of these sequences revealed that although the methylation-filtered libraries were sequenced at low coverage, they still tagged at least 66% of the RefSeq supported genes in the BAC and had a filtration power of at least 2.0. A total 33,752 microsatellites and 40,820 high-quality single nucleotide polymorphism (SNP) markers were identified. These represent the most comprehensive collection of microsatellites and SNPs to date and would be an important resource for genetic mapping and association studies. The gene models predicted from the assembled contigs were mined for genes of interest, and 242, 65 and 14 oil palm transcription factors, resistance genes and miRNAs were identified respectively. Examples of the transcriptional factors tagged include those associated with floral development and tissue culture, such as homeodomain proteins, MADS, Squamosa and Apetala2. The E. guineensis and E. oleifera hypomethylated sequences provide an important resource to understand the molecular mechanisms associated with important agronomic traits in oil palm.

Comparative genomic and transcriptomic analysis of selected fatty acid biosynthesis genes and CNL disease resistance genes in oil palm

Comparative genomics and transcriptomic analyses were performed on two agronomically important groups of genes from oil palm versus other major crop species and the model organism, Arabidopsis thaliana. The first analysis was of two gene families with key roles in regulation of oil quality and in particular the accumulation of oleic acid, namely stearoyl ACP desaturases (SAD) and acyl-acyl carrier protein (ACP) thioesterases (FAT). In both cases, these were found to be large gene families with complex expression profiles across a wide range of tissue types and developmental stages. The detailed classification of the oil palm SAD and FAT genes has enabled the updating of the latest version of the oil palm gene model. The second analysis focused on disease resistance (R) genes in order to elucidate possible candidates for breeding of pathogen tolerance/resistance. Ortholog analysis showed that 141 out of the 210 putative oil palm R genes had homologs in banana and rice. These genes formed 37 clusters with 634 orthologous genes. Classification of the 141 oil palm R genes showed that the genes belong to the Kinase (7), CNL (95), MLO-like (8), RLK (3) and Others (28) categories. The CNL R genes formed eight clusters. Expression data for selected R genes also identified potential candidates for breeding of disease resistance traits. Furthermore, these findings can provide information about the species evolution as well as the identification of agronomically important genes in oil palm and other major crops.

In silico characterization and expression profiling of the diacylglycerol acyltransferase gene family (DGAT1, DGAT2, DGAT3 and WS/DGAT) from oil palm, elaeis guineensis

The diacylglycerol acyltransferases (DGAT) (diacylglycerol:acyl-CoA acyltransferase, EC 2.3.1.20) are a key group of enzymes that catalyse the final and usually the most important rate-limiting step of triacylglycerol biosynthesis in plants and other organisms. Genes encoding four distinct functional families of DGAT enzymes have been characterised in the genome of the African oil palm, Elaeis guineensis. The contrasting features of the various isoforms within the four families of DGAT genes, namely DGAT1, DGAT2, DGAT3 and WS/DGAT are presented both in the oil palm itself and, for comparative purposes, in 12 other oil crop or model/related plants, namely Arabidopsis thaliana, Brachypodium distachyon, Brassica napus, Elaeis oleifera, Glycine max, Gossypium hirsutum, Helianthus annuus, Musa acuminata, Oryza sativa, Phoenix dactylifera, Sorghum bicolor, and Zea mays. The oil palm genome contains respectively three, two, two and two distinctly expressed functional copies of the DGAT1, DGAT2, DGAT3 and WS/DGAT genes. Phylogenetic analyses of the four DGAT families showed that the E. guineensis genes tend to cluster with sequences from P. dactylifera and M. acuminata rather than with other members of the Commelinid monocots group, such as the Poales which include the major cereal crops such as rice and maize. Comparison of the predicted DGAT protein sequences with other animal and plant DGATs was consistent with the E. guineensis DGAT1 being ER located with its active site facing the lumen while DGAT2, although also ER located, had a predicted cytosol-facing active site. In contrast, DGAT3 and some (but not all) WS/DGAT in E. guineensis are predicted to be soluble, cytosolic enzymes. Evaluation of E. guineensis DGAT gene expression in different tissues and developmental stages suggests that the four DGAT groups have distinctive physiological roles and are particularly prominent in developmental processes relating to reproduction, such as flowering, and in fruit/seed formation especially in the mesocarp and endosperm tissues.